The immune system is capable of producing two types of antigen-specific responses to foreign antigens. Cell-mediated immunity is the term used to refer to effector functions of the immune system mediated by T lymphocytes. Humoral immunity is the term used to refer to production of antigen-specific antibodies by B lymphocytes. It has long been appreciated that the development of humoral immunity against most antigens requires not only antibody-producing B lymphocytes but also the involvement of helper T (hereinafter Th) lymphocytes. (Mitchison, Eur. J. Immunol., 1:18-25 (1971); Claman and Chaperon, Transplant Rev., 1:92-119 (1969); Katz et al., Proc. Natl. Acad. Sci. USA, 70:2624-2629 (1973); Raff et al., Nature, 226:1257-1260 (1970)). Certain signals, or "help", are provided by Th cells in response to stimulation by Thymus-dependent (hereinafter TD) antigens. While some B lymphocyte help is mediated by soluble molecules released by Th cells (for instance lymphokines such as IL-4 and IL-5), activation of B cells also requires a contact-dependent interaction between B cells and Th cells. (Hirohata et al., J. Immunol., 140:3736-3744 (1988); Bartlett et al., J. Immunol., 143:1745-1765 (1989)). This indicates that B cell activation involves an obligatory interaction between cell surface molecules on B cells and Th cells. Such an interaction is further supported by the observation that isolated plasma membranes of activated T cells can provide helper functions necessary for B cell activation. (Brian, Proc. Natl. Acad. Sci. USA, 85:564-568 (1988); Hodgkin et al., J. Immunol., 145:2025-2034 (1990); Noelle et al., J. Immunol., 146:1118-1124 (1991)).
It is further known that in a contact-dependent process termed "T cell helper function", CD4.sup.+ T lymphocytes direct the activation and differentiation of B lymphocytes and thereby regulate the humoral immune response by modulating the specificity, secretion and isotype-encoded functions of antibody molecules (Mitchell et al., J. Exp. Med., 128:821 (1968); Mitchison, Eur. J. Immunol., 1:68 (1971); White et al., J. Exp. Med., 14:664 (1978); Reinherz et al., Proc. Natl. Acad. Sci. USA, 74:4061 (1979); Janeway et al., Immunol. Rev., 101:39 (1988); O'Brien et al. J. Immunol., 141:3335 (1988); Rahemtulla et al., Nature, 353:180 (1991); and Grusby et al., Science, 253:1417 (1991)).
The process by which T cells help B cells to differentiate has been divided into two distinct phases; the inductive and effector phases (Vitetta et al., Adv. Immunol., 45:1 (1989); Noelle et al., Immunol. Today, 11:361 (1990)). In the inductive phase, resting T cells contact antigen-primed B cells and this association allows clonotypic T cell receptor (TCR)-CD4 complexes to interact with Ia/Ag complexes on B cells (Janeway et al., Immunol. Rev., 101:39 (1988); Katz et al., Proc. Natl. Acad. Sci., 70:2624 (1973); Zinkernagel, Adv. Exp. Med., 66:527 (1976); Sprent, J. Exp. Med., 147:1159 (1978); Sprent, Immunol. Rev., 42:158 (1978); Jones et al., Nature, 292:547 (1981);
Julius et al., Eur. J. Immunol., 18:375 (1982); Chestnut et al., J. Immunol., 126:1575 (1981); and Rogozinski et al., J. Immunol., 126:735 (1984)). TCR/CD4 recognition of Ia/Ag results in the formation of stable T-B cognate pairs and bi-directional T and B cell activation (Sanders et al., J. Immunol., 137:2395 (1986); Snow et al., J. Immunol., 130:614 (1983); Krusemeier et al., J. Immunol., 140:367 (1988); Noelle et al., J. Immunol., 143:1807 (1989); Bartlett et al., J. Immunol., 143:1745 (1989); and Kupfer et al., Annu. Rev. Immunol., 7:309 (1987)). In the effector phase, activated T cells drive B cell differentiation by secreting lymphokines (Thompson et al., J. Immunol., 134:369 (1985)) and by contact-dependent stimuli (Noelle et al., J. Immunol., 143:1807 (1989); Clement et al., J. Immunol., 140:3736 (1984); Crow et al., J. Exp. Med., 164:1760 (1986); Brian, Proc. Natl. Acad. Sci., USA, 85:564 (1988); Hirohata et al., J. Immunol. 140:3736 (1988); Jover et al., Clin. Immunol. Immun., 53:90 (1989); Whalen et al., J. Immunol., 141:2230 (1988); Pollok et al., J. Immunol., 146:1633 (1991); and Bartlett et al., J. Immunol., 143:1745 (1990)), both of which are required for T cells to drive small resting B cells to terminally differentiate into Ig secreting cells (Clement et al., J. Immunol., 132:740 (1984); Martinez et al., Nature, 290:60 (1981); and Andersson et al., Proc. Natl. Acad. Sci., USA, 77:1612 (1980)).
Although the inductive phase of T cell help is Ag-dependent and MHC-restricted (Janeway et al., Immun. Rev., 101:34 (1988); Katz et al., Proc. Natl. Acad. Sci., USA, 10:2624 (1973); Zinkernagle, Adv. Exp. Med. Biol., 66:527 (1976)); the effector phase of T cell helper function can be Ag-independent and MHC-nonrestricted (Clement et al., J. Immunol., 132:740 (1984); Hirohata et al., J. Immunol., 140:3736 (1988); Whalen et al., J. Immunol., 143:1715 (1988)). An additional contrasting feature is that the inductive phase of T cell help often requires CD4 molecules and is inhibited by anti-CD4 mAb (Rogozinski et al., J. Immunol., 126:735 (1984)), whereas helper effector function does not require CD4 molecules (Friedman et al., Cell Immunol., 103:105 (1986)) and is not inhibited by anti-CD4 mAbs (Brian, Proc. Natl. Acad. Sci., USA, 85:564 (1988); Hirohata et al., J. Immunol., 140:3736 (1988); Whalen et al., J. Immunol., 143:1745 (1988); and Tohma et al., J. Immunol., 146:2547 (1991)). The non-specific helper effector function is believed to be focused on specific B cell targets by the localized nature of the T-B cell interactions with antigen specific, cognate pairs (Bartlett et al., J. Immunol., 143:1745 (1989); Kupfer et al., J. Exp. Med., 165:1565 (1987) and Poo et al., Nature, 332:378 (1988)).
Although terminal B cell differentiation requires both contact- and lymphokine-mediated stimuli from T cells, intermediate stages of B cell differentiation can be induced by activated T cell surfaces in the absence of secreted factors (Crow et al., J. Exp. Med., 164:1760 (1986); Brian, Proc. Natl. Acad. Sci., USA, 85:564 (1988); Sekita et al., Eur. J. Immunol., 18:1405 (1988); Hodgkin et al., J. Immunol., 145:2025 (1990); Noelle et al., FASEB J, 5:2770 (1991)). These intermediate effects on B cells include induction of surface CD23 expression (Crow et al., Cell Immunol., 121:94 (1989)), enzymes associated with cell cycle progression (Pollok et al., J. Immunol., 146:1633 (1991)) and responsiveness to lymphokines (Noelle et al., FASEB J, 5:2770 (1989); Pollok et al., J. Immunol., 146:1633 (1991)). Recently some of the activation-induced T cell surface molecules that direct B cell activation have been identified. Additionally, functional studies have characterized some features of activation-induced T cell surface molecules that direct B cell activation. First, T cells acquire the ability to stimulate B cells 4-8 h following activation (Bartlett et al., J. Immunol., 145:3956 (1990) and Tohma et al., J. Immunol., 146:2544 (1991)). Second, the B cell stimulatory activity associated with the surfaces of activated T cells is preserved on paraformaldehyde fixed cells (Noelle et al., J. Immunol., 143:1807 (1989); Cros et al., J. Exp. Med., 164:1760 (1986); Pollok et al., J. Immunol., 146:1633 (1991); Tohma et al., J. Immunol., 146:2544 (1991); and Kubota et al., Immunol., 72:40 (1991)) and on purified membrane fragments (Hodgkin et al., J. Immunol., 145:2025 (1990) and Martinez et al., Nature, 290:60 (1981)). Third, the B cell stimulatory activity is sensitive to protease treatment (Noelle et al., J. Immunol., 143:1807 (1989); Sekita et al., Eur. J. Immunol., 18:1405 (1988); and Hodgkin et al., J. Immunol., 145:2025 (1990). Fourth, the process of acquiring these surface active structures following T cell activation is inhibited by cycloheximide (Tohma et al., J. Immunol., 196:2349 (1991) and Hodgkin et al., J. Immunol., 195:2025 (1990) ).
A cell surface molecule, CD40, has been identified on immature and mature B lymphocytes which, when crosslinked by antibodies, induces B cell proliferation. Valle et al., Eur. J. Immunol., 19:1463-1467 (1989); Gordon et al., J. Immunol., 140:1425-1430 (1988); Gruder et al., J. Immunol., 142:4144-4152 (1989).
CD40 has been molecularly cloned and characterized (Stamenkovic et al., EMBO J., 8:1403-1410 (1989)).
CD40 is expressed on B cells, interdigitating dendritic cells, macrophages, follicular dendritic cells, and thymic epithelium (Clark, Tissue Antigens 36:33 (1990); Alderson et al., J. Exp. Med., 178:669 (1993); Galy et al., J. Immunol. 142:772 (1992) ). Human CD40 is a type I membrane protein of 50 kDa and belongs to the nerve growth factor receptor family (Hollenbaugh et al., Immunol. Rev., 138:23 (1994)). Signaling through CD40 in the presence of IL-10 induces IgA, IgM and IgG production, indicating that isotype switching is regulated through these interactions. The interaction between CD40 and its ligand results in a primed state of the B cell, rendering it receptive to subsequent signals.
Also, a ligand for CD40, gp39 (also called CD40 ligand or CD40L) has recently been molecularly cloned and characterized (Armitage et al., Nature, 357:80-82 (1992); Lederman et al., J. Exp. Med., 175:1091-1101 (1992); Hollenbaugh et al., EMBO J., 11:4313-4319 (1992)). The gp39 protein is expressed on activated, but not resting, CD4.sup.+ Th cells. Spriggs et al., J. Exp. Med., 176:1543-1550 (1992); Lane et al., Eur. J. Immunol., 22:2573-2578 (1992); and Roy et al., J. Immunol., 151:1-14 (1993). Cells transfected with gp39 gene and expressing the gp39 protein on their surface can trigger B cell proliferation and, together with other stimulatory signals, can induce antibody production. Armitage et al., Nature, 357:80-82 (1992); and Hollenbaugh et al., EMBO J., 11:4313-4319 (1992). In particular, the ligand for CD40, gp39, has been identified for the mouse (Noelle et al., Proc. Natl. Acad. Sci. USA, 89:6550 (1992); Armitage et al., Nature, 357:80 (1992)) and for humans (Hollenbaugh et al., Embo. J. 11:4313 (1992); Spriggs et al., J. Exp. Met., 176:1543 (1992)). gp39 is a type II membrane protein and is part of a new gene super family which includes TNF-.alpha., TNF-.beta. and the ligands for FAS, CD27, CD30 and 4-1BB.
Expression of gp39 can be readily induced in vitro on CD4.sup.+ T cells using either anti-CD3 antibody or phorbol myristate acetate (PMA) plus ionomycin. Expression is rapid and transient, peaking at 6-8 hours and returning to near resting levels between 24 and 48 hours (Roy et al., J. Immunol., 151:2497 (1993)). In vivo, gp39 has been reported in humans to be present on CD4.sup.+ T cells in the mantle and centrocytic zones of lymphoid follicles and the periarteriolar lymphocyte sheath of the spleen, in association with CD40.sup.+ B cells (Lederman et al., J. Immunol., 149:3807 (1992)). gp39.sup.+ T cells produce IL-2, IL-4 and IFN-.gamma. (Van der Eetwegh et al., J. Exp. Med., 178:1555 (1993)).
Unique insights into the novel role of gp39 in the regulation of humoral immunity have been provided by studies of a human disease, X-linked hyper-IgM syndrome (HIM). HIM is a profound, X-linked immunodeficiency typified by a loss in thymus dependent humoral immunity, the inability to produce IgG, IgA and IgE. Mutations in the gp39 gene were responsible for the expression of a non-functional gp39 protein and the inability of the helper T cells from HIM patients to activate B cells (Allen et al., Science, 259:990 (1993); Aruffo et al., Cell, 72:291 (1993); DiSanto et al., Nature, 361:541 (1993); Korthauer et al., Nature, 361:539 (1993)). These studies support the conclusion that early after T cell receptor engagement of the peptide/MHC class II complex, gp39 is induced on the cognate helper T cell, and the binding of gp39 to CD40 on the B cell induces the B cell to move into the cell cycle and differentiate to immunoglobulin (Ig) secretion and isotype switching.
Functional studies have shown that treatment of mice with anti-gp39 completely abolished the antibody response against thymus dependent antigens (SRBC and TNP-KLH), but not thymus independent antigens (TNP-Ficoll) (Foy et al., J. Exp. Med., 178:1567 (1993)). In addition, treatment with anti-gp39 prevented the development of collagen-induced arthritis (CIA) in mice injected with collagen (Durie et al., Science, 261:1328 (1993)). Finally, anti-gp39 prevented formation of memory B cells and germinal centers in mouse spleen (Foy et al., J. Exp. Med., 180:157 (1994)). Collectively, these data provide extensive evidence that the interaction between gp39 on T cells and CD40 on B cells is essential for antibody responses against thymus dependent antigens.
Recently, a number of murine models of autoimmune disease have been exploited to evaluate the potential therapeutic value of anti-gp39 administration on the development of disease. A brief discussion of the results of studies in these models are provided below:
Collagen-Induced Arthritis: CIA is an animal model for the human autoimmune disease rheumatoid arthritis (RA) (Trenthorn et al., J. Exp. Med., 146:857 (1977)). This disease can be induced in many species by the administration of heterologous type II collagen (Courtenay et al., Nature, 283:665 (1980); Cathcart et al., Lab. Invest., 54:26 (1986)).
To study the effect anti-gp39 on the induction of CIA (Durie et al., Science, 261:1328 (1993)) male DBA1/J mice were injected intradermally with chick type II collagen emulsified in complete Freund's adjuvant at the base of the tail. A subsequent challenge was carried out 21 days later. Mice were then treated with the relevant control antibody or anti-gp39. Groups of mice treated with anti-gp39 showed no titers of anti-collagen antibodies compared to immunized, untreated control mice. Histological analysis indicated that mice treated with anti-gp39 antibody showed no signs of inflammation or any of the typical pathohistological manifestations of the disease observed in immunized animals. These results indicated that gp39-CD40 interactions are absolutely essential in the induction of CIA. If the initial cognate interaction between the T cell and B cell is not obtained, then the downstream processes, such as autoantibody formation and the resulting inflammatory responses, do not occur.
Recently it has been shown that gp39 is important in activating monocytes to produce TNF-.alpha. and IL-6 in the absence of GM-CSF, IL-3 and IFN-.gamma. (Alderson et al., J. Exp. Med., 178:669 (1993)). TNF-.alpha. has been implicated in the CIA disease process (Thorbecke et al., Eur. J. Immunol., 89:7375 (1992) and in RA (DiGiovane et al., Ann. Rheum. Dis., 47:68 (1988); Chu et al., Arthrit. Rheum., 39:1125 (1991); Brennan et al., Eur. J. Immunol., 22:1907 (1992). Thus, inhibition of TNF-.alpha. by anti-gp39 may have profound anti-inflammatory effects in the joints of arthritic mice. Both inhibition of TNF-.alpha. and of T cell-B cell interactions by anti-gp39 may be contributory to manifestations of CIA.
Experimental Allergic Encephalomyelitis (EAE): EAE is an experimental autoimmune disease of the central nervous system (CNS) (Zamvil et al, Ann. Rev. Immunol., 8:579 (1990) and is a disease model for the human autoimmune condition, multiple sclerosis (MS) (Alvord et al., "Experimental Allergic Model for Multiple Sclerosis," NY 511 (1984)). It is readily induced in mammalian species by immunizations of myelin basic protein purified from the CNS or an encephalitogenic proteolipid (PLP). SJL/J mice are a susceptible strain of mice (H-2.sup.s) and, upon induction of EAE, these mice develop an acute paralytic disease and an acute cellular infiltrate is identifiable within the CNS.
Classen and co-workers (unpublished data) have studied the effects of anti-gp39 on the induction of EAE in SJL/J mice. They found that EAE development was completely suppressed in the anti-gp39 treated animals. In addition, anti-PLP antibody responses were delayed and reduced compared to those obtained for control animals.
EAE is an example of a cell-mediated autoimmune disease mediated via T cells, with no direct evidence for the requirement for autoantibodies in disease progression. Interference with the interaction between gp39 and CD40 prevents disease induction and the adoptive transfer of disease.
Chronic (c) and acute (a) graft-versus-host-disease (GVHD): Chronic and acute GVHD result from donor cells responding to host disparate MHC alleles. In cGVHD (H-2.sup.d .fwdarw.H-2.sup.bd), heightened polyclonal immunoglobulin production is due to the interaction of allospecific helper T cells and the host B cells. In vivo administration of anti-gp39 antibody blocked cGVHD-induced serum anti-DNA autoantibodies, IgE production, spontaneous immunoglobulin production in vitro, associated splenomegaly and the ability to transfer disease. Durie F. H. et al., J. Clin. Invest., 94:133 (1994). Antibody production remained inhibited for extended periods of time after termination of anti-gp39 administration. Anti-allogeneic cytotoxic T lymphocyte (CTL) responses induced in aGVHD were also prevented by the in vivo administration of anti-gp39. These data suggest that CD40-gp39 interactions are critical in the generation of both forms of GVHD. The fact that CTL responses were inhibited and a brief treatment with anti-gp39 resulted in long-term prevention of disease suggest permanent alterations in the T cell compartment by the co-administration of allogeneic cells and anti-gp39 antibody.
Various research groups have reported the production of murine antibodies specific to gp39, which are disclosed to possess therapeutic utility as immunosuppressants. For example, WO 93/09812, published May 27, 1993, and assigned to Columbia University; EP 0,555,880, published Aug. 18, 1993, and PCT US/94/09872, filed Sep. 2, 1994 by Noelle et al and assigned to Dartmouth College, describe murine antibodies specific to gp39 and their use as therapeutics.
However, while murine antibodies have applicability as therapeutic agents in humans, they are disadvantageous in some respects. Specifically, murine antibodies, because of the fact that they are of foreign species origin, may be immunogenic in humans. This often results in a neutralizing antibody response, which is particularly problematic if the antibodies are desired to be administered repeatedly, e.g., in treatment of a chronic or recurrent disease condition. Also, because they contain murine constant domains they may not exhibit human effector functions.
In an effort to eliminate or reduce such problems, chimeric antibodies have been disclosed. Chimeric antibodies contain portions of two different antibodies, typically of two different species. Generally, such antibodies contain human constant and another species, typically murine variable regions. For example, some mouse/human chimeric antibodies have been reported which exhibit binding characteristics of the parental mouse antibody, and effector functions associated with the human constant region. See, e.g., Cabilly et al., U.S. Pat. No. 4,816,567; Shoemaker et al., U.S. Pat. No. 4,978,745; Beavers et al., U.S. Pat. No. 4,975,369; and Boss et al., U.S. Pat. No. 4,816,397, all of which are incorporated by reference herein. Generally, these chimeric antibodies are constructed by preparing a genomic gene library from DNA extracted from pre-existing murine hybridomas (Nishimura et al., Cancer Research, 47:999 (1987)). The library is then screened for variable region genes from both heavy and light chains exhibiting the correct antibody fragment rearrangement patterns. Alternatively, cDNA libraries are prepared from RNA extracted from the hybridomas and screened, or the variable regions are obtained by polymerase chain reaction. The cloned variable region genes are then ligated into an expression vector containing cloned cassettes of the appropriate heavy or light chain human constant region gene. The chimeric genes are then expressed in a cell line of choice, usually a murine myeloma line. Such chimeric antibodies have been used in human therapy.
In a commonly assigned application, Ser. No. 07/912,292, "Primatized".TM. antibodies are disclosed which contain human constant and Old World monkey variable regions. These Primatized.TM. antibodies are well tolerated in humans given their low or weak immunogenicity.
Also, humanized antibodies are known in the art. Ideally, "humanization" results in an antibody that is less immunogenic, with complete retention of the antigen-binding properties of the original molecule. In order to retain all the antigen-binding properties of the original antibody, the structure of its combining-site has to be faithfully reproduced in the "humanized" version. This can potentially be achieved by transplanting the combining site of the nonhuman antibody onto a human framework, either (a) by grafting the entire nonhuman variable domains onto human constant regions to generate a chimeric antibody (Morrison et al., Proc. Natl. Acad. Sci., USA, 81:6801 (1984); Morrison and Oi, Adv. Immunol., 44:65 (1988) (which preserves the ligand-binding properties, but which also retains the immunogenicity of the nonhuman variable domains); (b) by grafting only the nonhuman CDRs onto human framework and constant regions with or without retention of critical framework residues (Jones et al., Nature, 321:522 (1986); Verhoeyen et al., Science, 239:1539 (1988)); or (c) by transplanting the entire nonhuman variable domains (to preserve ligand-binding properties) but also "cloaking" them with a human-like surface through judicious replacement of exposed residues (to reduce antigenicity) (Padlan, Molec. Immunol., 28:489 (1991)).
Essentially, humanization by CDR grafting involves transplanting only the CDRs onto human fragment onto human framework and constant regions. Theoretically, this should substantially eliminate immunogenicity (except if allotypic or idiotypic differences exist). However, it has been reported that some framework residues of the original antibody also need to be preserved (Riechmann et al., Nature, 332:323 (1988); Queen et al., Proc. Natl. Acad. Sci. USA, 86:10,029 (1989)).
The framework residues which need to be preserved can be identified by computer modeling. Alternatively, critical framework residues may potentially be identified by comparing known antibody combining site structures (Padlan, Molec. Immun., 31(3):169-217 (1994)).
The residues which potentially affect antigen binding fall into several groups. The first group comprises residues that are contiguous with the combining site surface which could therefore make direct contact with antigens. They include the amino-terminal residues and those adjacent to the CDRs. The second group includes residues that could alter the structure or relative alignment of the CDRs either by contacting the CDRs or the opposite chains. The third group comprises amino acids with buried side chains that could influence the structural integrity of the variable domains. The residues in these groups are usually found in the same positions (Padlan, 1994 (Id.) according to the adopted numbering system (see Kabat et al., "Sequences of proteins of immunological interest, 5th ed., Pub. No. 91-3242, U.S. Dept. Health & Human Services, NIH, Bethesda, Md., 1991).
However, while humanized antibodies are desirable because of their potential low immunogenicity in humans, their production is unpredictable. For example, sequence modification of antibodies may result in substantial or even total loss of antigen binding function, or loss of binding specificity. Alternatively, "humanized antibodies" may still exhibit immunogenicity in humans, irrespective of sequence modification.
Thus, there still exists a significant need in the art for novel humanized antibodies to desired antigens. More specifically, there exists a need in the art for humanized antibodies specific to gp39, because of their potential as immunotherapeutic agents.